Fig 2: BAP1 function relies on its deubiquitinase activity against H2AK119ub1. a Reintroduction of wild-type or catalytically dead (C91S) BAP1 in BAP1 KO cells. Top panel: western blot analysis of BAP1 expression in cytoplasmic (C) or nuclear (N) fractions. HDAC1 is used as a loading control. Bottom panel: RT-qPCR analysis of three BAP1-regulated genes in wild-type, BAP1 KO and the two rescue conditions. Horizontal bars indicate the mean expression. n = 3. b Western blot analysis of RING1A, RING1B, BAP1, H2AK119ub1, and H3K27me3 in wild-type, RING1A/B double KO, or RING1A/B; BAP1 triple KO HAP1 cells. H4 is used as a loading control. c Venn diagram showing the overlap between genes downregulated in BAP1 KO or upregulated in RING1A/B dKO cells. d Box-plots (median, lower, and upper quartiles, lowest and highest values) of log2 transcript per million (TPM) expression values of BAP1-only-regulated genes (top) and BAP1/PRC1-regulated genes61 in WT and RING1A/B dKO cells. P-value of the Mann–Whitney test on WT versus RING1A/B dKO comparison is indicated. e Scatterplot showing log2 fold-change (logFC) expression between RING1A/B dKO and RING1A/B; BAP1 tKO cells as a function of average log2 counts per million (logCPM). BAP1, the only differentially expressed genes is highlighted in purple. f RT-qPCR analysis of CYP26A1, RARB, DHRS3, and FAM46A expression following RA treatment at different time-points in wild-type or BAP1, RING1A/B, RING1A/B;BAP1 KO cells

Fig 3: Rnf2-null zebrafish mutant displayed severe cardiac defects. (A) The target site (sequence highlighted in blue) is located in exon 3. The PAM site is underlined and highlighted in red; (B) DNA sequencing identified that two mutant alleles carried a 5 bp (rnf2f5) and 8 bp (rnf2f8) deletion respectively. The deletion sites are indicated by red arrows; (C) Schematic diagram of the wild-type and mutant Rnf2 proteins. Zebrafish wild-type Rnf2 contained an N-terminal Ring-finger domain (yellow) and a C-terminal RAWUL domain. The two mutant proteins were truncated before the RING-finger domain; (D) The rnf2−/− larvae displayed severe pericardia edema. The hearts are indicated by white arrows, the stringy heart in rnf2−/− is depicted by white dotted lines, scale bar: 0.2 mm; (E) Scatter plot showing the sectional area of edema in wild-type (black) and rnf2−/− (red). (F) Histological sections of 4 dpf heart, boxes in yellow indicate the AVC and valves, white boxes indicate the bulbous arteriosus, scale bar: 50 μm, n(WT) = 2, n (rnf2−/−) = 3; (G) Western blot verified the deletion of Rnf2 protein. Gapdh was set as internal reference; (H) Bar graph showing the percentage of embryos with edema. **, p < 0.01; ***, p < 0.001; n, sample number.

Fig 4: Comparison of BAP1 and the CREBBP and SMARCB1 co-activator proteins. a RT-qPCR analysis of CYP26A1 and RARB expression following RA treatment at different time-points in wild-type or BAP1, CREBBP, and SMARCB1 KO cells. n = 2. b Violin plots showing log2 fold-change expression of RA-responsive genes (n = 114 genes, see text for details) in wild-type and BAP1 KO cells. P-value from the Mann–Whitney test is shown. c Violin plots showing log2TPM expression of BAP1- CREBBP-, and SMARCB1-regulated genes in wild-type (WT) or in the respective KO conditions. d Venn diagram showing overlap between genes that are downregulated in BAP1, CREBBP, and SMARCB1 KO cells. e Heatmaps showing ASXL1 and RNA PolII density around the TSS and termination end site (TES) (including 2-kb upstream and downstream) scaled to an equivalent 10 kb in human HEK-293 cells. Corresponding average profiles are plotted above each heatmap. f Scatterplot showing PolII versus ASXL1 enrichment around the TSS (including 2 kb upstream and downstream) at all annotated genes. Pearson correlation coefficient is displayed. g Snapshots of ASXL1 and RNA PolII enrichment at representative regions. The input is displayed below each corresponding ChIP-seq experiment

Fig 5: BAP1.com promotes transcription independently of an antagonism with the PRC2 complex. a Heatmaps showing H2AK119ub1, H3K27me3, and H3K4me3 distribution in a −5/ + 5 kb window around the transcription start site (TSS) of genes downregulated in BAP1 KO cells in wild-type and BAP1 KO cells. Corresponding average profiles are plotted on top of each heatmap. b Western blot analysis of BAP1 and EZH2 in single and double BAP1/EZH2 KO cells. HDAC1 is used as a loading control. c Dot plot showing proliferation of wild-type cells and two independent clones of BAP1/EZH2 dKO cells. n = 3. d Heatmap of gene expression (Z-scores) in the different genotypes. Top: BAP1-only-regulated genes, and bottom: BAP1- and EZH2-regulated genes, TSS = transcription start site. e Box-plots (median, lower, and upper quartiles, lowest and highest values) of log2 transcript per million (TPM) expression values of BAP1-only-regulated genes (top) and BAP1/EZH2-regulated genes61 in the different conditions as indicated. Result of the Mann–Whitney test on the EZH2 KO versus wild-type comparison is indicated. f Plot showing average enrichment of H2AK119ub1, H3K27me3, and H3K4me3 in a −5/ + 5 kb around the TSS for BAP1-only-regulated genes (top) and BAP1/EZH2-regulated genes61. g Example snapshots of H2AK119ub1, H3K27me3, and H3K4me3 enrichment in WT and BAP1 KO cells at a BAP1-only-regulated gene and a BAP1/EZH2-regulated gene (middle). Expression values of the corresponding genes across WT, EZH2, BAP1, and BAP1/EZH2 KO conditions as detected in corresponding RNA-seq data are shown on the right. Horizontal bars indicate the mean expression